Tài liệu Báo cáo Y học: Systematic search for zinc-binding proteins in Escherichia coli potx

Systematic search for zinc-binding proteins in Escherichia coli

Akira Katayama1,2, Atsuko Tsujii2

, Akira Wada3

, Takeshi Nishino2 and Akira Ishihama

1

National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka, Japan; 2

Nippon Medical School, Department

of Biochemistry and Molecular Biology, Bunkyo-ku, Tokyo, Japan; 3

Osaka Medical School, Department of Physics, Takatsuki,

Osaka, Japan

A systematic search for Escherichia coli proteins with the

zinc-binding activity was performed using the assay of

radioactive Zn(II) binding to total E. coli proteins fraction￾ated by two methods of two-dimensional gel electrophoresis.

A total of 30–40 radioactive spots were identified, of which

14 have been assigned from N-terminal sequencing.In

addition to five known zinc-binding proteins, nine zinc￾binding proteins were newly identified including: acetate

kinase (AckA), DnaK, serine hydroxymethyltransferase

(GlyA), transketolase isozymes (TktA/TktB), translation

elongation factor Ts (Tsf), ribosomal proteins L2 (RplB),

L13 (RplM) and one of S15 (RpsO), S16 (RpsP) or S17

(RpsQ).Together with about 20 known zinc-binding pro￾teins, the total number of zinc-binding proteins in E. coli

increased up to more than 30 species (or more than 3% of

about 1000 proteins expressed under laboratory culture

conditions).The specificity and affinity of zinc-binding were

analysed for some of the zinc-binding proteins.

Keywords: zinc-binding protein; Escherichia coli; proteome;

two-dimensional gel electrophoresis.

Zinc is an essential trace element, but virtually nontoxic, in

contrast to iron, copper and mercury.Over 300 enzymes or

proteins have been identified that require zinc for function

[1,2].Physical and chemical properties of zinc, such as its

stable association with proteins and its co-ordination

flexibility, make it highly adaptable to meeting the needs

of proteins and enzymes that carry out diverse biological

functions [3].In zinc-containing enzymes or proteins, zinc

has two major functions, i.e. catalytic and structural. The

catalytic role specifies that zinc participates directly in

enzyme catalysis, while structural zinc atoms are required

for stabilization of proteins by supporting their folding and

oligomerization.Zinc is therefore not simply the cofactor

for enzyme catalytic functions but also the structural factor

for folding of domains involved in protein–protein and

protein–DNA interactions.

A large majority of the zinc-containing enzymes have a

single zinc site consisting of a combination of specific

amino-acid residues such as Cys, His, Asp and Glu, and a

solvent water molecule completing the co-ordination sphere

[3].After the finding of a number of zinc-containing DNA￾binding proteins in higher eukaryotes, many different types

of the zinc-binding motif have been identified, including

those tetrahedrally co-ordinated to imidazole nitrogen

atoms from His and thiol groups from Cys [2].The

functions of protein-bound zinc are beginning to catch up

with the increasing number of zinc-containing proteins.Up

to the present time, the structural and functional roles of

zinc have been analysed in detail with zinc-containing

proteins from higher eukaryotes, but little is known about

the zinc-binding proteins in prokaryotes.Ros homologues

that exit in plant-associated agrobacteria are the only

bacterial proteins with the typical C2H2-type zinc-finger

motif [4].Various types of zinc-containing protein with the

zinc finger or zinc cluster exist in yeast, which, however,

lacks proteins with the hormone receptor-type zinc-binding

motif.This type of zinc-binding proteins appears in

Caenorhabditis elegans [5] and the number of this type of

zinc-binding proteins increases in higher animals.

The aim of this study is to identify as many zinc-binding

proteins as possible in the model prokaryote, Escherichia

coli.For a systematic and experimental detection of zinc￾binding proteins, we employed a conventional method of

radioactive zinc blotting with whole cell extracts fraction￾ated by two methods of two-dimensional gel electrophor￾esis, the widely used O’Farrell system [6] and the newly

developed radical-free and highly reducing (RFHR) method

[7].Results indicate that most of the newly identified

bacterial zinc-binding proteins do not contain the known

zinc-binding motifs, most of which have been identified in

higher eukaryotes.This report also describes the affinity and

specificity of zinc binding for some of the E. coli zinc￾binding proteins.

MATERIALS AND METHODS

Preparation of cell extracts

E. coli W3110 A-type [8] was grown in Luria–Bertani

medium until late exponential phase (D600 ¼ 0.8 ). Cells

were harvested by centrifugation and cell extracts were

prepared according to Iwakura et al.[9].In brief, cells were

suspended in lysis buffer (50 mM Tris/HCl, pH 8.0 at 4
C,

1 mM EDTA) and lysozyme and phenylmethanesulfonyl

fluoride were added to the final concentration of 1 mgÆmL)1

and 1 mM, respectively.After incubation for 10 min at

Correspondence to A.Ishihama, National Institute of Genetics,

Department of Molecular Genetics, Mishima, Shizuoka 411-8540,

Japan.Fax: + 81 559 81 6746, Tel.: + 81 559 81 6741,

E-mail: [email protected]

Abbreviations: PVDF, poly(vinylidene difluoride); RFHR, radical-free

and highly reducing.

(Received 16 July 2001, revised 28 February 2002,

accepted 22 March 2002)

Eur. J. Biochem. 269, 2403–2413 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.02900.x